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Topical application of the hexane fraction of Lacistema pubescens reduces skin inflammation and cytokine production in animal model Josiane Mello da Silva a , Jéssica Leiras Mota Conegundes a , Renata de Freitas Mendes a , Nícolas de Castro Campos Pinto a , Ana Cristina Moura Gualberto b , Antônia Ribeiro a , Jacy Gameiro b , Jair Adriano Kopke de Aguiar c , Maria Christina Marques Nogueira Castañon d and Elita Scio a a Laboratory of Bioactive Natural Products, Department of Biochemistry, b Department of Parasitology, Microbiology and Immunology, Biological Sciences Institute, c Glycoconjugate Analysis Laboratory, Department of Biochemistry, d Department of Morphology, Biological Sciences Institute, Federal University of Juiz de Fora, Juiz de Fora, MG, Brazil Keywords cytokine; dermatoxicity; Lacistema pubescens; myeloperoxidase; skin inflammation Correspondence Elita Scio, Laboratory of Bioactive Natural Products, Department of Biochemistry, Federal University of Juiz de Fora, 36036 900 Juiz de Fora, MG, Brazil. E-mail: [email protected] Received February 5, 2015 Accepted June 21, 2015 doi: 10.1111/jphp.12463 Abstract Objectives The aim of this study was to investigate the acute topical anti- inflammatory effect of the hexane fraction (HLP) of Lacistema pubescens in mice. Methods Ear oedema models induced by croton oil, arachidonic acid, phenol, histamine, ethyl phenyl propiolate and capsaicin. Histopathological analyses of ear tissue samples sensitized with croton oil were performed. Myeloperoxidase activity (MPO), the pro-inflammatory cytokine-inhibitory effect and dermatoxicity were also evaluated. Key findings HLP (1, 0.5 and 0.1 mg/ear) resulted in a substantial reduction in skin thickness or tissue weight on all models tested, except for capsaicin-induced ear oedema, similar to dexamethasone (0.1 mg/ear) and/or indomethacin (0.5 mg/ear). Histopathological analyses and neutrophil-mediated MPO activity confirmed the topical anti-inflammatory effect of HLP. In addition, HLP reduced IL-1β, IL-6 and tumour necrosis factor-α cytokine levels. Sitosterol-rich fraction (SRF), obtained from HLP fractionation, reduced ear oedema on croton oil and phenol models at the same dose of dexamethasone (0.1 mg/ear). No dermotoxicity was observed. Conclusions The mechanism of action of HLP was associated with the inhibition of several pro-inflammatory mediators, including cytokines, arachidonic acid metabolites and histamine, which suggested a glucocorticoid-like effect, reinforced by the presence of the steroid sitosterol. This is the first report on anti- inflammatory activity of L. pubescens leaves. Introduction The skin is the body organ responsible for a direct interac- tion between the environment and the organism, and its main function is to form an effective barrier to protect the organism from several external stimuli. Thus, as a mecha- nism of defence, the skin is able to recognize, discriminate and integrate specific signals from the environment and generate appropriate responses to maintain the body homeostasis. [1] Normally, this defence mechanism does not cause serious damage; however, an inappropriate or misdi- rected immune activity can implicate in the pathogenesis of a large variety of inflammatory skin disorders. [2] For example, the most common inflammatory dermatosis are psoriasis and atopic dermatitis, which have a high impact on the life quality, as psychological, self-esteem and body image disturbances. [3] It is widely recognized that the modulation of the synthe- sis of inflammatory mediators may be used therapeutically against skin inflammations. [4] For this purpose, skin diseases can be treated either topically or systemically with glucocorticoids, antihistamines, non-steroidal And Pharmacology Journal of Pharmacy Research Paper © 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 1613–1622 1613
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Topical application of the hexane fraction of Lacistemapubescens reduces skin inflammation and cytokineproduction in animal modelJosiane Mello da Silvaa, Jéssica Leiras Mota Conegundesa, Renata de Freitas Mendesa,Nícolas de Castro Campos Pintoa, Ana Cristina Moura Gualbertob, Antônia Ribeiroa, Jacy Gameirob,Jair Adriano Kopke de Aguiarc, Maria Christina Marques Nogueira Castañond and Elita Scioa

aLaboratory of Bioactive Natural Products, Department of Biochemistry, bDepartment of Parasitology, Microbiology and Immunology, BiologicalSciences Institute, cGlycoconjugate Analysis Laboratory, Department of Biochemistry, dDepartment of Morphology, Biological Sciences Institute,Federal University of Juiz de Fora, Juiz de Fora, MG, Brazil

Keywordscytokine; dermatoxicity; Lacistema pubescens;myeloperoxidase; skin inflammation

CorrespondenceElita Scio, Laboratory of Bioactive NaturalProducts, Department of Biochemistry, FederalUniversity of Juiz de Fora, 36036 900 Juiz deFora, MG, Brazil.E-mail: [email protected]

Received February 5, 2015Accepted June 21, 2015

doi: 10.1111/jphp.12463

Abstract

Objectives The aim of this study was to investigate the acute topical anti-inflammatory effect of the hexane fraction (HLP) of Lacistema pubescens in mice.Methods Ear oedema models induced by croton oil, arachidonic acid, phenol,histamine, ethyl phenyl propiolate and capsaicin. Histopathological analyses of eartissue samples sensitized with croton oil were performed. Myeloperoxidase activity(MPO), the pro-inflammatory cytokine-inhibitory effect and dermatoxicity werealso evaluated.Key findings HLP (1, 0.5 and 0.1 mg/ear) resulted in a substantial reduction inskin thickness or tissue weight on all models tested, except for capsaicin-inducedear oedema, similar to dexamethasone (0.1 mg/ear) and/or indomethacin(0.5 mg/ear). Histopathological analyses and neutrophil-mediated MPO activityconfirmed the topical anti-inflammatory effect of HLP. In addition, HLP reducedIL-1β, IL-6 and tumour necrosis factor-α cytokine levels. Sitosterol-rich fraction(SRF), obtained from HLP fractionation, reduced ear oedema on croton oiland phenol models at the same dose of dexamethasone (0.1 mg/ear). Nodermotoxicity was observed.Conclusions The mechanism of action of HLP was associated with the inhibitionof several pro-inflammatory mediators, including cytokines, arachidonic acidmetabolites and histamine, which suggested a glucocorticoid-like effect, reinforcedby the presence of the steroid sitosterol. This is the first report on anti-inflammatory activity of L. pubescens leaves.

Introduction

The skin is the body organ responsible for a direct interac-tion between the environment and the organism, and itsmain function is to form an effective barrier to protect theorganism from several external stimuli. Thus, as a mecha-nism of defence, the skin is able to recognize, discriminateand integrate specific signals from the environment andgenerate appropriate responses to maintain the bodyhomeostasis.[1] Normally, this defence mechanism does notcause serious damage; however, an inappropriate or misdi-rected immune activity can implicate in the pathogenesis of

a large variety of inflammatory skin disorders.[2] Forexample, the most common inflammatory dermatosis arepsoriasis and atopic dermatitis, which have a high impacton the life quality, as psychological, self-esteem and bodyimage disturbances.[3]

It is widely recognized that the modulation of the synthe-sis of inflammatory mediators may be used therapeuticallyagainst skin inflammations.[4] For this purpose, skindiseases can be treated either topically or systemicallywith glucocorticoids, antihistamines, non-steroidal

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And PharmacologyJournal of Pharmacy

Research Paper

© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 1613–1622 1613

anti-inflammatory drugs, and with monoclonal antibodiesand recombinant cytokines.[5] However, these therapeuticalternatives are usually aggressive and not effective in allcases, limiting their use.[6] As alternative, extracts and iso-lated compounds from herbal medicine have been studiedto discover new effective and safe topical anti-inflammatorydrugs.[7]

Lacistema pubescens Mart. (Lacistemataceae), popularlyknown as ‘canela-vermelha’[8] is a native tree from Brazil,also found in other countries of South America. Species ofthe genus Lacistema have been traditionally used for someethnopharmacological purposes mainly by indigenouspeoples of Brazil and Peru to combat rheumatism, vomit-ing, dysentery, fever and body aches.[9–11]

Recently, pharmacological studies performed by ourgroup reported some preliminary results indicating apotential in-vivo topical anti-inflammatory activity usingcroton oil-induced ear oedema model of the hexane frac-tion (HLP) of L. pubescens leaves. Compounds like tocoph-erol, sitosterol, phytol among others were identified in thehexane extract using gas chromatography and massspectrometry (GC-MS). This study also reported theantinociceptive effect of this species.[12] Furthermore,a potential antioxidant,[13] antileishmanial and anti-proliferative activities[14] of the crude methanol extract ofthe leaves and its fractions were reported.

The aim of this study was to confirm, using differentirritants agents, the preliminary anti-inflammatoryactivity previously observed for the HLP of the leaves ofL. pubescens[12] and its possible mechanism of action. Asitosterol-rich fraction (SRF) obtained from chromato-graphic fractionation of HLP was also evaluated toverify whether this compound contributed for the anti-inflammatory activity observed for HLP.

Materials and Methods

Plant material

L. pubescens leaves were collected in Juiz de Fora, MinasGerais, Brazil, in December 2011. A voucher specimen(CESJ 49751) has been deposited at the Leopoldo KriegerHerbarium of the Federal University of Juiz de Fora.

Extraction and fractionation

Preparation of the HLP was previously described indetail.[12] Fractionation of HLP was performed by the fol-lowing manner: HLP (6 g) was chromatographed on a 42 ×5 cm column contained silica-gel (70–230 mesh) with a gra-dient of hexane-ethyl acetate (EtOAc) (90:10 v/v – 100%EtOAc) and EtOAc-MeOH (90:10 v/v – 100% MeOH) toobtain a total of 17 fractions (F1–F17). Fraction F9(802 mg) was rechromatographed and eluted with hexane-

EtOAc (90:10 v/v to 20:80 v/v) and EtOAc-MeOH (90:10v/v to 50:50 v/v) to obtain 12 fractions (FF1–FF12).

Chemical analysis

The chemical constitutions of the fractions were analyzedby GC-MS[12] by computer comparison of the mass spectrawith those in the Wiley and NIST libraries, mass fragmenta-tion and retention indices in reference to an n-alkane seriesin a temperature-programmed run.[15] High pressure liquidchromatography (HPLC) analysis was performed forHLP.[14] Detection was performed at 210 nm. Sitosterolstandard (Sigma-Aldrich, Saint Louis, MO, USA) was usedin this experiment as marker under the same conditionsused for HLP.

Animals

Male Swiss mice (Mus musculus) weighting 25–35 g andmale Wistar rats (Rattus norvegicus) weighing 160–200 g,obtained from the Reproduction Biology Center of theFederal University of Juiz de Fora, were housed in a roomkept under controlled conditions at 23 ± 2°C and on a 12 hlight/12 h dark cycle. They were provided with standardpellets and tap water ad libitum. Throughout the experi-ments, animals were processed according to the ethicalguidelines for the care of laboratory animals. The study wasapproved by the Brazilian College of Animal Experimenta-tion (COBEA-protocols n° 021/2012 and 013/2013).Animals were divided in groups of six to eight animals each.

Acute dermal irritation/corrosion test

The acute dermal irritation/corrosion study was carried outin accordance with the OECD Guideline 404[16] with minormodifications. Six rats with intact skin were assigned to twotreatment groups: control and HLP. On day 0 of the testperiod, hair was shaved from the back of each rat and HLP(0.5 g) applied to a small area of the skin. After a 4-h expo-sure period, animals were examined for signs of erythemaand oedema at grading intervals of 60 min, and then at 24,48 and 72 h.

Croton oil-induced mouse ear oedema

Oedema was induced on the right ear by topical applicationof 20 μl of croton oil 5% (v/v) in acetone. After 15 min,HLP (1, 0.5 and 0.1 mg/ear), SRF (0.1 mg/ear), dexametha-sone (0.1 mg/ear, used as reference drug) or vehicle acetonewas applied topically on the right ear, whereas the left ear ofall animals received 20 μl of vehicle. The thickness of theears was measured before and 6 h after induction of inflam-mation.[17] After 24 h, animals treated with HLP wereeuthanized and ear punch biopsies were collected and sub-jected to histopathological analysis or were snap frozen in

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liquid nitrogen and stored at −80°C until further processedfor myeloperoxidase (MPO) determination.

Arachidonic acid (AA), phenol, capsaicin andethyl phenylpropiolate (EPP)-induced mouseear oedema

Inflammation was induced applying on the inner surface ofthe right ear 20 μl of the following irritants diluted inacetone: AA 2 mg/ml, 10% phenol (v/v), capsaicin 0.02 mg/μl, and EPP 50 mg/ml. After 15 min, 20 μl of HLP (1, 0.5and 0.1 mg/ear), indomethacin (0.5 mg/ear, used as a posi-tive control for AA) or dexamethasone (0.1 mg/ear, used asa positive control for phenol and EPP) or SRF (0.1 mg/ear –only phenol model) was applied topically on the right ear,whereas the left ear received 20 μl of vehicle. The earoedema was evaluated 30 min after capsaicin, 1 h after AAand EPP, and 2 h after phenol application.[18–22]

Subcutaneous histamine-induced mouseear oedema

Right ears were treated topically with HLP (1, 0.5, 0.1 mg/ear), vehicle (20 μl/ear) or dexamethasone (0.1 mg/ear).After 15 min, the oedema was induced in by intradermalapplication of 10 μl of histamine dihydrochloride (0.1 mg/ml) dissolved in 0.9% saline, whereas the left ear wasinjected with 10 μl of 0.9% saline. Ear oedema was evalu-ated 2 h after histamine application.[23]

Ear oedema measurement

Oedema was expressed as increase in ear weight (allmodels) or ear thickness variation (croton oil), which wasmeasured using a digital micrometre. To evaluate the earweight, animals were euthanized, 6-mm diameter of earbiopsies were collected using a metal punch, and the biop-sies were individually weighed on a precision balance. Theextent of the oedema was expressed as the differencebetween the weight of the section removed from the rightear (which received the irritant agent) and the weight of thesection obtained from the left ear (which received vehicleused to dilute the irritant agent). The mean oedema inhibi-tion percentage (%) was calculated using the followingformula: inhibition (%) = 100 − ((A × 100)/B), where ‘A’ isthe mean of oedema weight (mg) of the group treated withHLP, indomethacin or dexamethasone, and ‘B’ is themean of oedema weight of the untreated group (negativecontrol).

MPO determination

Ear neutrophil infiltration was quantified by measuringmyeloperoxidase (MPO) activity as described previously.[24]

The results were expressed as specific activity (mUE/mgprotein).

Cytocines determination

Quantitative cytokine assays were performed by standardcapture enzyme-linked immunosorbent assay (ELISA). Theears were collected, minced, homogenized in extractionbuffer 0.5% bovine serum albumin (BSA), 0.1 Mphenylmethylsulphonyl fluoride, 0.1 M benzethonium chlo-ride, 10 mM ethylenediaminetetracetic acid and 20 kIU/mlaprotinin and centrifuged at 10 000 g for 15 min at 4°C. Thesupernatants were stored and used to dosage of IL-1β,tumour necrosis factor (TNF)-α, IL-6 using commercialELISA kits according to the procedures supplied by themanufacturer (Life Technologies do Brasil Ltda Alto dePinheiros, São Paulo, SP, Brazil). The levels of the cytokineproteins were determined in duplicate by an ELISA readerat 450 nm.

Histopathology

Ear biopsies from croton oil-induced ear oedema were col-lected and fixed in 70% ethanol for 24 h and then preservedin 10% formalin. Subsequently, the ears were dehydrated,blocked in paraffin and then sectioned with a microtome(4 μm). The cross-sections were stained with haematoxylinand eosin for the evaluation of histopathological changesrelated to acute inflammatory process. A representative areawas selected for qualitative light microscope analysis (100×and 400× magnification).

Statistical analysis

The results were expressed as mean ± standard error ofmean (SEM). The comparison between groups was assessedby ANOVA followed by Student–Newman–Keuls test usingthe software GraphPad Prism 5.0 (San Diego, CA, USA).Values of P < 0.05 were considered statistically significantcompared with negative control.

Results

Chemical analysis

The HPLC chromatogram profile of the HLP was per-formed and the presence of sitosterol was confirmed byexternal standard (Figure 1). GC-MS analysis of the frac-tions identified FF3 as sitosterol-rich fraction with 70%purity, named in this work as SRF.

Dermatoxicity test

It was assessed a possible acute dermal irritation of HLPwhen administered on skin. No dermal responses, includingerythema/eschar or oedema, were found in rats treated withHLP during the evaluation period.

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© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 1613–1622 1615

Effect of HLP on croton oil, AA, phenol,histamine, EPP and capsaicin-induced miceear oedema

As demonstrated in Figure 2a, HLP in all doses tested (1, 0.5and 0.1 mg/ear) significantly reduced the ear oedema 6 hafter topical application of croton oil when compared withthe group treated with acetone (negative control). As apositive control, dexamethasone (0.1 mg/ear) significantlyreduced the oedema (inhibition of 72%) compared withnegative control group. The calculated inhibition to HLPwas 74%, 67% and 65% at a dose of 1, 0.5 and 0.1 mg/earrespectively (P < 0.001). HLP also caused significantoedema reduction against AA (inhibition of 74% and 70%at 1.0 and 0.5 mg/ear, respectively, P < 0.001) (Figure 2b),phenol (inhibition of 95%, 50% and 81% at 1, 0.5 and0.1 mg/ear, respectively, P < 0.001) (Figure 2c), histamine(inhibition of 64% at 1.0 mg/ear, P < 0.001) (Figure 2d) andEPP (inhibition of 67% and 66% at 1 and 0.5 mg/ear,respectively, P < 0.05) (Figure 2e). In contrast, the topicalapplication of HLP did not present significant reduction oncapsaicin-induced ear inflammation (P > 0.05) (Figure 2f).

Effect of HLP on myeloperoxidase activity

Myeloperoxidase activity was measured in the ear punchbiopsies taken 24 h after oil croton administration. AsFigure 3 shown, the MPO activity of the negative controlgroup was significantly increased. However, treatment withboth dexamethasone and HLP produced a remarkable inhi-bition of MPO activity. Dexamethasone at 0.1 mg/ear andHLP at 1, 0.5 and 0.1 mg/ear doses inhibited cell infiltration

by 49%, 58%, 51% and 54% (P < 0.01, Figure 3). This inhi-bition was confirmed by histopathological analysis.

Effect of HLP on pro-inflammatorycytokines production

The pro-inflammatory cytokine-inhibitory effect of HLPwas investigated of the ear punch biopsies taken 6 h after oilcroton administration to assess their effectiveness at themolecular level. As shown in Figure 4, topical application ofcroton oil caused an increase in the production of IL-1β(A), IL-6 (B) and TNF-α (C) 6 h after challenge. In con-trast, treatment with HLP or dexamethasone reduced IL-1β(P < 0.05), IL-6 (P < 0.001) and TNF-α (P < 0.05) cytokineslevels significantly.

Effect of SRF on croton oil andphenol-induced mice ear oedema

To investigate whether SRF contributed for the anti-inflammatory activity observed for HLP, we employedcroton oil and phenol-induced mice ear oedema consider-ing the most significative anti-inflammatory effect of HLPon these models. As demonstrated in Figure 5a, SRF in thesame dose of dexamethasone (0.1 mg/ear), significantlyreduced the ear oedema (inhibition of 65%), 6 h aftertopical application of croton oil when compared with thenegative control group (P < 0.001). As demonstrated inFigure 5b, SRF caused significant oedema reduction on sen-sitized ears with phenol, in the same dose of the referencedrug (0.1 mg/ear), showing an inhibition effect of 70%(P < 0.05).

5 10 15 20

18.5

25Retention time (min)

250

300

200

150

100

50

0

Ab

sorb

ance

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AU

)

Figure 1 High-pressure liquid chromatography chromatogram of Lacistema pubescens hexane fraction. Peak identified: sitosterol (retentiontime = 18.5 min). Hexane fraction conditions: Zorbax SB-18 (250 mm × 4.6 mm i.d.; 5 μm); methanol-acetonitrile (70:30 v/v); 1.0 ml/min; injectionvolume, 20 μl; UV detection at 210 nm.

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Histopathology analysis

Histopathological analysis of ear tissue 24 h after croton oiltreatment revealed a significant increase in the dermisthickness associated a vasodilatation, oedema and markedinfiltration of inflammatory cells associated (Figure 6b)when compared with non-inflamed ear (Figure 6a, vehicleacetone). The ears treated with dexamethasone (Figure 6c)and HLP at all doses (Figure 6d–f) demonstrated a decreasein these inflammatory parameters when compared with theears that received croton oil and vehicle.

Discussion

Previous studies from our group have showed that HLP ofL. pubescens leaves possesses in-vivo topical anti-inflammatory activity using croton oil-induced ear oedemamodel; however, ear oedema models induced by differentirritant agents allow the proposition of the possible mecha-nism of action. Besides, they promote conditions thatresemble some types of dermatitis observed in humans.[20]

The present work provides evidence that HLP clearlyexerted anti-inflammatory effect on all models tested,

Croton oil

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Figure 2 Effect of hexane fraction (HLP) on croton oil (a), arachidonic acid (AA) (b), phenol (c), histamine (d), ethyl phenylpropiolate (EPP) (e) andcapsaicin (f) induced mice ear oedema. Dexamethasone (DEX) and Indomethacin (INDO) were used as positive control. Statistical analysis: one-wayANOVA followed by Newman–Keuls test (n = 6–8). *P < 0.05, **P < 0.01, ***P < 0.001 compared with negative control group (C). ##Statisticallyequal to reference drug.

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except for capsaicin-induced ear oedema, in a mannersimilar to dexamethasone and indomethacin. Furthermore,the acute dermal irritation study of the HLP showed nodermal responses, such as erythema or oedema.

Croton oil contains 12-o-tetracanoilphorbol-13-acetate(TPA) and other phorbol esters as main irritant agents, andonce topically applied, results in an increased vascular per-meability, migration of polymorphonuclear leucocytes(mainly neutrophils), liberation of histamine and serotonin,and a moderate synthesis of eicosanoids.[25] Croton oil isable to activate protein kinase C (PKC), which activatesother enzymatic cascades in turn, such as mitogen-activatedprotein kinases and phospholipase A2 (PLA2), leading tothe release of platelet activation factor and AA. Thesemetabolites, as well as cytokines, are mediators of inflam-matory pathways and are responsible for triggering andmaintaining inflammation.[26] Thus, as croton oil induces an

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Figure 3 Effect of hexane fraction (HLP) on production ofmyeloperoxidase (MPO). Dexamethasone (DEX) was used as referencedrug. Statistical analysis: oneway ANOVA followed by Newman–Keulstest (n = 6–8). **P < 0.01 compared with negative control group (C).##Statistically equal to reference drug.

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Figure 4 Effect of hexane fraction (HLP – 1.0 mg/ear) on production of IL-1β (a), IL-6 (b) and tumour necrosis factor-α (c). Dexamethasone (DEX –0.1 mg/ear) was used as reference drug. Statistical analysis: one-way ANOVA followed by Newman–Keuls test (n = 6–8). *P < 0.05, ***P < 0.001compared with negative control group (C). ##Statistically equal to reference drug.

DEXC SRF DEXC SRF

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Figure 5 Effect of sitosterol-rich fraction (SRF – 0.1 mg/ear) on croton oil (a) and phenol (b) induced mice ear oedema. Dexamethasone (DEX –0.1 mg/ear) was used as reference drug. Statistical analysis: one-way ANOVA followed by Newman–Keuls test (n = 6–8). *P < 0.05, **P < 0.01,***P < 0.001 compared with negative control group (C). ##Statistically equal to reference drug.

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(a) (b)

(c) (d)

(e) (f)

Figure 6 Photomicrograph of transverse sections of mice ears sensitized with topical application of croton oil 5% (v/v) in acetone (b–f) or vehicleacetone (a, not inflamed), (haematoxylin and eosin 100× e 400×). Treatments: acetone (b), dexamethasone 0.1 mg/ear (c), HLP 1.0 mg/ear (d)0.5 mg/ear (e) and 0.1 mg/ear (f). Keys indicate epidermis, and arrows indicate leukocyte infiltration in the dermis. The shown sections are repre-sentative of five animals per group.

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inflammatory process by activating several pathways, cyclo-oxygenases (COX) and lipo-oxygenases (5-LOX) inhibitors,leukotriene B4 (LTB4) antagonists and corticosteroidspresent topical anti-inflammatory action in this model.[27]

As described, HLP evidenced a significant decrease on earoedema at all doses tested (Figure 2a).

AA is a precursor of inflammatory eicosanoids such asprostaglandin E2 and leukotrienes (produced via COX-1,COX-2 and 5-LOX enzymes). Indomethacin is related to thenon-selective inhibition of the isoforms of COX and clearlyreversed the oedema induced by AA.[20] Dexamethasone,likewise, modulates the production of AA metabolites,probably via inhibition of COX and LOX enzymes synthe-sis.[28] HLP 1 and 0.5 mg/ear decreased the ear oedema inthis model (Figure 2b).

Phenol-induced ear oedema is an appropriate model forsimulating contact dermatitis. When phenol is topicalapplied, keratinocytes produce chemical mediators that areimportant in primary contact irritation responses, includ-ing cytokines, such as IL-1α, TNF-α and IL-8.[29] Thismechanism is independent of PKC activation pathway,which in turn results in the release of inflammatory media-tors such as AA metabolites and reactive oxygen species.[30]

HLP significantly reduced the phenol-induced oedema at alldoses, suggesting a probable activity against contact derma-titis (Figure 2c). This activity may be related to the influ-ence on the production of AA metabolites and cytokines.

HLP markedly inhibited the histamine (Figure 2d) andEPP (Figure 2e) ear induced oedema. Histamine causesvasodilation and an increase in microvascular permeabil-ity[31] as well as stimulates nerve fibres. Antihistamines andcorticosteroids reduce the oedema in this model, which isinvolved in immediate type allergic reactions.[32] The localinflammatory response by EPP is mainly vascular innature,[33] involving the release of several inflammatorymediators, such as histamine, serotonin, bradykinin andprostaglandins.[34] On the other hand, capsaicin when incontacts with epidermis exerts an immediate effect on aTRPV1 receptor target and stimulates a neurogenic inflam-matory response.[20] HLP did not present significantoedema reduction in this model, suggesting that it does notinterfere in substances or receptors involved in capsaicin-activated inflammatory pathways.

Neutrophils are key players in the recognition and elimi-nation of pathogens, but their improper activation isthought to induce tissue lesions and contribute to thepathophysiology of various inflammatory diseases.[35] MPOis known as a direct marker of neutrophil infiltration; there-fore, its activity inhibition can be used as an indicator ofanti-inflammatory action.[36] HLP was able to reduce thenumber of leukocytes in the inflamed tissue evidenced byMPO activity and by the histopathological analysis, whichsuggests a possible interference in cell migration during the

inflammatory process. The histopathological analysis alsoconfirmed that HLP markedly inhibited the oedema inten-sity (Figure 6).

This study also showed that topical exposure to crotonoil resulted in an increased secretion of TNF-α, IL-6 andIL-1β in mouse ear biopsy homogenates. IL-1β, a pro-inflammatory cytokine, in which main sources are thekeratinocytes in the skin, activates neutrophils, monocytes,eosinophils and basophils.[37] IL-1β is important in anumber of severe inflammatory diseases and most of thesediseases can be completely controlled by anti-IL-1β treat-ment.[38] IL-6 is involved in the growth and differentiationof dermal and epidermal cells, and acts as a chemotacticfactor for T cells.[39] TNF-α is also an important cytokineinvolved in the maintenance of inflammatory processes inthe skin, and TNF-α is stressed by its capacity to induceIL-1β and IL-6.[40] HLP (1 mg/ear) significantly decreasedthe levels of these cytokines (Figure 4), suggesting that thismechanism, at least partially, contributed to its anti-inflammatory response in acute skin inflammation, consid-ering the results obtained from phenol topical application,which induces the production of these inflammatorymediators (Figure 2c).

Considering the most significative anti-inflammatoryeffect of HLP in croton oil- and phenol-induced earoedema, these models were selected to verify if SRF contrib-uted, in any manner, to the anti-inflammatory activityobserved for HLP. In fact, SRF evidenced a significantdecrease on ear oedema at the same dose of dexamethasone(0.1 mg/ear) in both models (Figure 5). Glucocorticoids,including sitosterol and dexamethasone, bind to specificintracellular receptors that activate or repress gene tran-scription to inhibit the actions of the immune system as awhole. Glucorticoids inhibit the expression of COX-2,iNOS, adhesion factors, complement factors and cytokines,and induce the expression of other proteins, such asanexin-1, which inhibits the prostanoids synthesis. Asdexamethasone, HLP inhibited the action of differentphlogistic agents, which induced inflammation by differentpathways, suggesting a glucocorticoid-like effect of HLP,which is reinforced by the presence of sitosterol.[41] Thesedata are also supported by the literature, which reportedthat sitosterol (dose of 0.5 mg/ear), isolated of Achilleaageratum, is effective as a topical anti-inflammatory agentin acute inflammatory process.[42]

Conclusions

In summary, the results of this study showed the effective-ness and safeness of HLP as a topical anti-inflammatoryagent that may serve as a source for developing effectivedrugs for skin inflammatory disorders. Furthermore, themechanism of action of HLP was associated with the

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inhibition of several pro-inflammatory mediators, includingcytokines, AA metabolites and histamine, which suggested aglucocorticoid-like effect, reinforced by the presence of thesteroid sitosterol. Further studies are necessary to identifyother chemical constituents that may be contributing to thisactivity. According to our knowledge, this is the first reporton anti-inflammatory activity of L. pubescens leaves.

Declarations

Conflict of interest

The Author(s) declare(s) that they have no conflicts ofinterest to disclose.

Funding

The authors are grateful to Fundação de Amparo a Pesquisado Estado de Minas Gerais (FAPEMIG) and UniversidadeFederal de Juiz de Fora (UFJF), Brazil, for financial support.

Acknowledgements

The authors thank Dr Fatima Regina Salimena for thebotanical identification of species and Delfino AntonioCampos for technical assistance.

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